PROJECT SUMMARY Despite making up only 2% of the US population, men who have sex with men (MSM) accounted for 67% of new HIV infections in 2014, and incidence rates among adolescent MSM are alarmingly high in some areas. One potential barrier to the effectiveness of biomedical HIV prevention interventions among MSM is the physiologic efficiency of HIV transmission across the rectal mucosa, where approximately 70% of infections are thought to occur among MSM, as compared to vaginal or penile transmission. However, the majority of HIV mucosal transmission research has concentrated on vaginal transmission in women and non-human primates, which is then extrapolated and applied to understanding the mechanisms of rectal transmission among MSM. The rectal mucosa, with its single layer of columnar epithelium, is theoretically more prone to mechanical trauma during sexual intercourse than the vagina or penis, and exposure to semen during coitus causes a pro- inflammatory cascade in the female genital tract that may have similar effects in the rectum. In our preliminary data with 41 adult HIV-negative MSM aged 18-45, we show a distinct innate and adaptive pro-inflammatory immune response to condomless receptive anal intercourse (CRAI) in the rectal mucosa, and that the diversity and composition of the rectal mucosal microbiota differ between adult MSM who engage in CRAI versus men who do not engage in anal intercourse (AI) with MSM engaging in CRAI being enriched for the family Prevotellaceae. In this application, we propose to expand our previous studies to examine how factors such as age and episodic, frequent, consensual sexual activity can influence rectal transmission, and to determine whether the mucosal inflammation associated with CRAI leads to delayed mucosal healing after injury. In Aim 1, we will compare the rectal mucosal immune milieu between adolescent MSM with recent anal sex debut, adult MSM who have engaged in CRAI for > 5 years, and men who do not engage in AI with state of the art flow cytometry, RNA-Seq, and microbiota sequencing assays and examine the ex vivo impact on HIV susceptibility with rectal explant challenge experiments. In Aim 2, we will tease out the effects of rectal mucosal trauma due to episodic anal intercourse with and without semen exposure in a cohort of adult MSM engaging in CRAI. Finally, in aim 3, we will examine whether adult MSM who engage in CRAI exhibit delayed rectal mucosal healing after experimentally induced injury with rectal biopsy during high resolution anoscopy with follow-up digital imaging. We will build upon our successful translational mucosal immunology program with a highly successful clinical research and retention infrastructure that was designed to understand factors that may influence rectal transmission among MSM. A better understanding of rectal HIV transmission among MSM will contribute valuable information to the development of biomedical HIV prevention interventions, including an HIV vaccine.